PROJECT SUMMARY Protein post-translational modifications are ubiquitous events that regulate the core biological functions of the cell. Lysine acetylation was first characterized in the context of chromatin structure and transcription, where abundant acetylation of histone proteins is known to regulate gene expression. Recent proteomic data have expanded this paradigm and suggest that lysine acetylation is ubiquitous among both nuclear and cytosolic proteins. Mechanistic studies to document how these marks are placed, utilized to transduce signals, and eliminated when signals need to be turned off have not kept pace with proteomic discoveries. A major bottleneck persists due to the paucity of transferrable chemical biology probes of acetyllysine that do not perturb structure, which is pre-requisite for downstream structure-mechanism studies. The PI?s laboratory is uniquely positioned to advance the acetylation signaling field through this proposal, which will seek to determine the technological feasibility of direct acetyllysine readout through NMR spectroscopy applied to 13C-enriched acetyl groups. The PI has a proven track record of technology development in 13C direct-detect biomolecular NMR, which is used to probe the biophysics of disordered proteins and the complexes they form. The current project proposes to generate acetyllysine with uniform 13C and 15N enrichment in order to leverage the similarity between the peptide bond and the side chain acetamide functional group. In this context, the first specific aim of this project is to develop a chemical biology route to site-specific isotopic-enrichment of acetyllysine. The initial strategy involves synthetic coupling of 13C-acetate to Coenzyme-A, which will be used as a cofactor for enzymatic transfer of the acetyl group to recombinant protein, with or without isotope labelling of the protein. To validate this procedure with a thoroughly described enzyme-substrate pair, the preliminary work will employ acetylation of Histone 3, Lysine 14 by the Ada2/Gcn5 complex. Because all known lysine acetyltransferases use acetyl-CoA as a cofactor for substrate acetylation, the developed approach will be general and readily adapted to a broad range of enzyme-substrate pairs. The second specific aim is to design broadly applicable NMR experiments to target acetyllysine with high specificity. This project will lead to 13C direct-detect (N-Acetyl)-COCme and (N- Acetyl)-CON experiments that will allow direct NMR readout of the acetyl group in a broader range of targets. Further, 3D spectroscopy built up from these new 2D detection platforms will be developed to unambiguously connect acetyl resonances with the aliphatic side chain. The new technology proposed here will enable applications including site-specific assignment of newly-identified modifications, investigation of novel binding modes as new reader proteins are discovered, and determination of solution NMR structures based on isotope filtered NOE measurement. The generality of the developed technology will provide sustained benefits for the transcription and signaling communities, potentially driving translation of biological discoveries involving the acetyllysine post-translational modification toward the clinic.